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Although antivirals are important tools to control severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, effective vaccines are essential to control the current coronavirus disease 2019 (COVID-19) pandemic. Plant-derived virus-like particle (VLP) vaccine candidates have previously demonstrated immunogenicity and efficacy against influenza. Here, we report the immunogenicity and protection induced in rhesus macaques by intramuscular injections of a VLP bearing a SARS-CoV-2 spike protein (CoVLP) vaccine candidate formulated with or without Adjuvant System 03 (AS03) or cytidine-phospho-guanosine (CpG) 1018. Although a single dose of the unadjuvanted CoVLP vaccine candidate stimulated humoral and cell-mediated immune responses, booster immunization (at 28 days after priming) and adjuvant administration significantly improved both responses, with higher immunogenicity and protection provided by the AS03-adjuvanted CoVLP. Fifteen micrograms of CoVLP adjuvanted with AS03 induced a polyfunctional interleukin-2 (IL-2)-driven response and IL-4 expression in CD4 T cells. Animals were challenged by multiple routes (i.e., intratracheal, intranasal, and ocular) with a total viral dose of 106 plaque-forming units of SARS-CoV-2. Lower viral replication in nasal swabs and bronchoalveolar lavage fluid (BALF) as well as fewer SARS-CoV-2-infected cells and immune cell infiltrates in the lungs concomitant with reduced levels of proinflammatory cytokines and chemotactic factors in the BALF were observed in animals immunized with the CoVLP adjuvanted with AS03. No clinical, pathologic, or virologic evidence of vaccine-associated enhanced disease was observed in vaccinated animals. The CoVLP adjuvanted with AS03 was therefore selected for vaccine development and clinical trials.

Several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines are being deployed, but the global need greatly exceeds the supply, and different formulations might be required for specific populations. Here we report Day 42 interim safety and immunogenicity data from an observer-blinded, dose escalation, randomized controlled study of a virus-like particle vaccine candidate produced in plants that displays the SARS-CoV-2 spike glycoprotein (CoVLP: NCT04450004). The co-primary outcomes were the short-term tolerability/safety and immunogenicity of CoVLP formulations assessed by neutralizing antibody (NAb) and cellular responses. Secondary outcomes in this ongoing study include safety and immunogenicity assessments up to 12 months after vaccination. Adults (18–55 years, n = 180) were randomized at two sites in Quebec, Canada, to receive two intramuscular doses of CoVLP (3.75 μg, 7.5 μg, and 15 μg) 21 d apart, alone or adjuvanted with AS03 or CpG1018. All formulations were well tolerated, and adverse events after vaccination were generally mild to moderate, transient and highest in the adjuvanted groups. There was no CoVLP dose effect on serum NAbs, but titers increased significantly with both adjuvants. After the second dose, NAbs in the CoVLP + AS03 groups were more than tenfold higher than titers in Coronavirus 2019 convalescent sera. Both spike protein-specific interferon-γ and interleukin-4 cellular responses were also induced. This pre-specified interim analysis supports further evaluation of the CoVLP vaccine candidate., Safety and immunogenicity results in humans of a two-dose SARS-CoV-2 vaccine made from plants support further assessment of potential efficacy.

The regulation of molecular farming is a complex topic because plants and plant-based systems are relative newcomers among the many production platforms available for recombinant proteins. The regulations specific for different types of product (human/veterinary pharmaceuticals and medical devices, cosmetics, diagnostics, and research reagents) must therefore be overlaid with the regulations governing hitherto unfamiliar production platforms, and this must be achieved in different jurisdictions that handle genetically modified organisms (and genetically modified plants in particular) in very different ways. This chapter uses examples of different product types and production methods in three different jurisdictions (the USA, the EU, and Canada) to demonstrate some of the challenges facing the regulatory authorities.

Backgroud: coronavirus-like particles (CoVLP) that are produced in plants and display the prefusion spike glycoprotein of the original strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are combined with an adjuvant (Adjuvant System 03 [AS03]) to form the candidate vaccine.Methods: in this phase 3, multinational, randomized, placebo-controlled trial conducted at 85 centers, we assigned adults (≥18 years of age) in a 1:1 ratio to receive two intramuscular injections of the CoVLP+AS03 vaccine or placebo 21 days apart. The primary objective of the trial was to determine the efficacy of the CoVLP+AS03 vaccine in preventing symptomatic coronavirus disease 2019 (Covid-19) beginning at least 7 days after the second injection, with the analysis performed after the detection of at least 160 cases.Results: a total of 24,141 volunteers participated in the trial; the median age of the participants was 29 years. Covid-19 was confirmed by polymerase-chain-reaction assay in 165 participants in the intention-to-treat population; all viral samples that could be sequenced contained variants of the original strain. Vaccine efficacy was 69.5% (95% confidence interval [CI], 56.7 to 78.8) against any symptomatic Covid-19 caused by five variants that were identified by sequencing. In a post hoc analysis, vaccine efficacy was 78.8% (95% CI, 55.8 to 90.8) against moderate-to-severe disease and 74.0% (95% CI, 62.1 to 82.5) among the participants who were seronegative at baseline. No severe cases of Covid-19 occurred in the vaccine group, in which the median viral load for breakthrough cases was lower than that in the placebo group by a factor of more than 100. Solicited adverse events were mostly mild or moderate and transient and were more frequent in the vaccine group than in the placebo group; local adverse events occurred in 92.3% and 45.5% of participants, respectively, and systemic adverse events in 87.3% and 65.0%. The incidence of unsolicited adverse events was similar in the two groups up to 21 days after each dose (22.7% and 20.4%) and from day 43 through day 201 (4.2% and 4.0%).Conclusions: the CoVLP+AS03 vaccine was effective in preventing Covid-19 caused by a spectrum of variants, with efficacy ranging from 69.5% against symptomatic infection to 78.8% against moderate-to-severe disease. (Funded by Medicago; ClinicalTrials.gov number, NCT04636697.).

Partial neutralization of the Golgi lumen pH by the ectopic expression of influenza virus M2 proton channel is useful to stabilize acid-labile recombinant proteins in plant cells, but the impact of pH gradient mitigation on host cellular functions has not been investigated. Here, we assessed the unintended effects of M2 expression on the leaf proteome of Nicotiana benthamiana infiltrated with the bacterial gene vector Agrobacterium tumefaciens. An isobaric tags for relative and absolute quantification quantitative proteomics procedure was followed to compare the leaf proteomes of plants agroinfiltrated with either an "empty" vector or an M2-encoding vector. Leaves infiltrated with the empty vector had a low soluble protein content compared to noninfiltrated control leaves, associated with increased levels of stress-related proteins but decreased levels of photosynthesis-associated proteins. M2 expression partly compromised these effects of agroinfiltration to restore soluble protein content in the leaf tissue, associated with restored levels of photosynthesis-associated proteins and reduced levels of stress-related proteins in the apoplast. These data illustrate the cell-wide influence of the Golgi lumen pH homeostasis on the leaf proteome of N. benthamiana responding to microbial challenge. They also underline the relevance of assessing the eventual unintended effects of accessory proteins used to modulate specific cellular or metabolic functions in plant protein biofactories.

Since 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection resulting in the coronavirus disease 2019 (COVID-19) has afflicted hundreds of millions of people in a worldwide pandemic. Several safe and effective COVID-19 vaccines are now available. However, the rapid emergence of variants and risk of viral escape from vaccine-induced immunity emphasize the need to develop broadly protective vaccines. A recombinant plant-derived virus-like particle vaccine for the ancestral COVID-19 (CoVLP) recently authorized by Canadian Health Authorities and a modified CoVLP.B1351 targeting the B.1.351 variant (both formulated with the adjuvant AS03) were assessed in homologous and heterologous prime-boost regimen in mice. Both strategies induced strong and broadly cross-reactive neutralizing antibody (NAb) responses against several Variants of Concern (VOCs), including B.1.351/Beta, B.1.1.7/Alpha, P.1/Gamma, B.1.617.2/Delta and B.1.1.529/Omicron strains. The neutralizing antibody (NAb) response was robust with both primary vaccination strategies and tended to be higher for almost all VOCs following the heterologous prime-boost regimen.

The binding of influenza hemagglutinin (HA) to sialic acid (SA) receptors plays a well-defined role in shaping infection but the impact of such binding on vaccine responses has not yet been explored. We generated a virus-like particle (VLP) vaccine bearing the HA of H1N1 A/California/07/09 that is unable to bind to its α(2,6)-linked SA receptor (H1

As the SARS-COV-2 pandemic evolves, what is expected of vaccines extends beyond efficacy to include consideration of both durability and variant cross-reactivity. This report expands on previously reported immunogenicity results from a Phase 1 trial of an AS03-adjuvanted, plant-based coronavirus-like particle (CoVLP) displaying the spike (S) glycoprotein of the ancestral SARS-CoV-2 virus in healthy adults 18-49 years of age (NCT04450004). When humoral and cellular responses against the ancestral strain were evaluated 6 months post-second dose (D201), 100% of vaccinated individuals retained binding antibodies, and ∼95% retained neutralizing antibodies; interferon gamma (IFN-γ) and interleukin 4 (IL-4) responses directed against the ancestral S protein were also still detectable in ∼94% and ∼92% of vaccinees respectively. Variant-specific, cross-reactive neutralizing antibody (NAb) levels were assessed at D42 and D201 using both live wild-type and pseudovirion assays (Alpha, Beta, Gamma) or the wild-type assay alone (Delta, Omicron). In the wild-type assay, broad cross-reactivity was detected against all variants at D42 (100% Alpha and Delta, 94% Beta and Gamma, 74% Omicron). At D201, cross-reactive antibodies were detectable in almost all participants against Alpha, Gamma and Delta variants (94%) and the Beta variant (83%) and in a smaller proportion against Omicron (44%). Results were similar in the pseudovirion assay (D42, 100% cross-reactivity to Alpha and Gamma variants, 95% to Beta variant, D201, 94% for Alpha, Beta and Gamma variants). These data suggest that two doses of 3.75 µg CoVLP+AS03 elicit a durable and cross-reactive response that persists for at least 6 months post-vaccination.

The rapid spread of SARS-CoV-2 continues to impact humanity on a global scale with rising total morbidity and mortality. Despite the development of several effective vaccines, new products are needed to supply ongoing demand and the needs of specific populations. We report herein a pre-specified interim analysis of the phase 2 portion of an ongoing Phase 2/3, randomized, placebo-controlled trial of a coronavirus virus-like particle (CoVLP) vaccine candidate produced in plants that displays the SARS-CoV-2 spike glycoprotein adjuvanted with AS03 (NCT04636697). A total of 753 subjects were recruited between 25 November 2020 and 24 March 2021 into three groups: Healthy Adults (18-64 years: N=306), Older Adults (≥ 65 years: N=282) and Adults with Comorbidities (≥18 years: N=165) and randomized 5:1 to receive two intramuscular doses of either vaccine CoVLP (3.75 μg/dose + AS03) or placebo 21 days apart. This report presents safety, tolerability and immunogenicity data collected up to 21 days after the second dose. The immune outcomes presented include neutralizing antibody (NAb) titres and cellular (IFN-γ and IL-4 ELISpot) responses. In this study, CoVLP+AS03 was well-tolerated and adverse events (AE) after each dose were generally mild to moderate and transient. Solicited AEs in Older Adults and Adults with Comorbidities were generally less frequent than in Healthy Adults. CoVLP+AS03 induced seroconversion in >35% of subjects in each group after the first dose and in ∼98% of subjects 21 days after the second dose. In all treatment groups, NAb levels were ∼10-fold higher than those in a panel of convalescent sera. A significant minority (∼20%) of subjects had evidence of a pre-existing IFN-γ response to the S protein and almost all subjects in all groups (>88%) had detectable cellular responses (IFN-γ, IL-4 or both) at 21 days after the second dose. A Th1-biased response was most evident after the first dose and was still present after dose two. These data demonstrated that CoVLP+AS03 will likely be well-tolerated and highly immunogenic in adults ≥18 years of age with and without comorbidities.

Although antivirals are important tools to control the SARS-CoV-2 infection, effective vaccines are essential to control the current pandemic. Plant-derived virus-like particle (VLP) vaccine candidates have previously demonstrated immunogenicity and efficacy against influenza. Here we report the immunogenicity and protection induced in macaques by intramuscular injections of VLP bearing SARS-CoV-2 spike protein (CoVLP) vaccine candidate formulated with or without Adjuvant System 03 (AS03) or cytosine phosphoguanine (CpG) 1018. Although a single dose of unadjuvanted CoVLP vaccine candidate stimulated humoral and cell-mediated immune responses, booster immunization (at 28 days after prime) and adjuvants significantly improved both responses with a higher immunogenicity and protection provided by AS03 adjuvanted CoVLP. Fifteen microgram CoVLP adjuvanted with AS03 induced a balanced IL-2 driven response along with IL-4 expression in CD4 T cells and mobilization of CD4 follicular helper cells (Tfh). Animals were challenged by multiple routes (i.e. intratracheal, intranasal and ocular) with a total viral dose of 106 plaque forming units of SARS-CoV-2. Lower viral replication in nasal swabs and broncho-alveolar lavage (BAL) as well as fewer SARS-CoV-2 infected cells and immune cell infiltrates in the lungs concomitant with reduced levels of pro-inflammatory cytokines and chemotactic factors in BAL were observed in the animals immunized with CoVLP adjuvanted with AS03. No clinical, pathologic or virologic evidences of vaccine associated enhanced disease (VAED) were observed in vaccinated animals. CoVLP adjuvanted with AS03 was therefore selected for vaccine development and clinical trials.

Background Virus-like particles (VLPs) are unable to replicate in the recipient but stimulate the immune system through recognition of repetitive subunits. Parenterally delivered rotavirus-VLP (Ro-VLP) vaccine could have the potential to overcome the weaknesses of licensed oral live-attenuated rotavirus vaccines, namely, low efficacy in low-income and high mortality settings and a potential risk of intussusception. Methods A monovalent Ro-VLP composed of viral protein (VP) 7, VP6 and VP2 of G1 genotype specificity was produced in Nicotiana benthamiana using Agrobacterium tumefaciens infiltration-based transient recombinant expression system. Plants expressing recombinant G1 Ro-VLP were harvested, then the resultant biomass was processed through a series of clarification and purification steps including standard extraction, filtration, ultrafiltration and chromatography. The purified G1 Ro-VLP was subsequently examined for its immunogenicity and toxicological profile using animal models. Results G1 Ro-VLP had a purity of ≥90% and was structurally similar to triple-layered rotavirus particles as determined by cryogenic transmission electron microscopy. Two doses of aluminum hydroxide-adjuvanted G1 Ro-VLP (1 μg, 5 μg or 30 μg), administered intramuscularly, elicited a robust homotypic neutralizing antibody response in rats. Also, rabbits administered G1 Ro-VLP (10 μg or 30 μg) four times intramuscularly with aluminum hydroxide adjuvant did not show any significant toxicity. Conclusions Plant-derived Ro-VLP composed of VP7, VP6 and VP2 structural proteins would be a plausible alternative to live-attenuated oral rotavirus vaccines currently distributed worldwide.

Longer AbstractBackgroundThe stabilized prefusion form of the SARS-CoV-2 spike protein is produced by transient expression in Nicotiana benthamiana. The trimeric spike glycoproteins are displayed at the surface of self-assembling Virus-Like-Particles that mimic the shape and the size of the virus. The candidate vaccine (CoVLP) administered alone or with AS03 or CpG1018 adjuvants was evaluated in a Phase 1 trial in healthy adults. (ClinicalTrials.gov number NCT04450004)MethodsThe study was a randomized, partially-blinded, prime-boost 21 days apart, dose-escalation Phase 1 study intended to assess the safety, tolerability, and immunogenicity of CoVLP at three dose levels (3.75 µg, 7.5 µg, and 15 µg) unadjuvanted or adjuvanted with either CpG 1018 or AS03 in 180 SARS-CoV-2 seronegative healthy adults 18 to 55 years of age. Enrollment was staggered for dose-escalation. At each dose level, the vaccine was initially administered to a small number of subjects. Vaccination of the remaining subjects at the same dose level and the next higher vaccine dose level was administered with approval of an Independent Data Monitoring Committee (IDMC). The same procedure was followed for the second vaccine administration. Monitoring of safety signals was performed throughout the study with pre-determined pausing/stopping rules if there was clear evidence of harmful effects such as severe adverse events (AEs) related to the treatment. The primary endpoints were the safety and tolerability of the vaccine after each dose and the immunogenicity as assessed by neutralizing antibody responses assessed using a vesicular stomatitis virus (VSV) pseudovirion assay and interferon-gamma (IFN-γ) and interleukin-4 (IL-4) ELISpot assays at Days 0, 21 and 42. Secondary endpoints were anti-spike antibody responses by ELISA and neutralizing antibodies measured by live virus plaque reduction neutralization test (PRNT) assay at Days 0, 21 and 42 and immunogenicity with additional safety and immunogenicity endpoints planned for 6-months following the last vaccination. The anti-spike and neutralizing antibody responses were compared with 23 convalescent serum samples from symptomatic Covid-19 patients. We performed a primary analysis at day 42.ResultsA total of 180 subjects (102 females: 78 males: average 34.3 years) were recruited to the study and interim safety and immunogenicity data up to day 42 after the first dose are reported here. There was no obvious CoVLP dose effect in safety outcomes for any of the formulations tested and all formulations were generally well-tolerated. Most solicited local and systemic AEs were mild-moderate and transient. Reactogenicity was increased in all adjuvanted formulations and was generally highest in the CoVLP+AS03 groups. Local and systemic adverse events were reported with similar frequency after the first and second doses in subjects who received either CoVLP alone or CoVLP+CpG1018 but increased in both frequency and severity after the second dose in the CoVLP+AS03 groups. CoVLP alone only elicited a weak total anti-spike IgG response at the highest dose level and little-to-no neutralization antibody response, even after the second dose. Cellular responses in the CoVLP alone groups (IFNγ and IL-4) were detectable after the second dose but were still only marginally above background levels. The addition of either adjuvant substantially increased both antibody and cellular responses at most CoVLP dose levels and changes were most pronounced after the second dose. However, a substantial neutralizing antibody response after the first dose was only seen in all CoVLP+AS03 groups. After the second dose, both total anti-spike IgG and neutralizing antibody titers in the CoVLP+AS03 groups were higher than those in the CoVLP+CpG1018 groups. The antibody titers achieved were either similar to (CoVLP+CpG1018) or at least 10-times higher (CoVLP+AS03) than those seen in convalescent plasma. Administration of CoVLP with either adjuvant also significantly increased the cellular responses. After 2 doses, both IFN-γ and IL-4 responses were significantly increased in the CoVLP+CpG1018 groups. In the CoVLP+AS03 groups, significant increases in the cellular responses were observed after the first dose while IFN-γ and IL-4 increased further in both magnitude and number of subjects responding after the second dose. Again, the cellular responses in the CoVLP+AS03 groups were higher than those seen in the CoVLP+CpG1018 groups.ConclusionThese data demonstrate that CoVLP administered with either CpG1018 or AS03 has a safety profile similar to other candidate vaccines for SARS-CoV-2. When administered with either AS03 or CpG1018, several of the CoVLP dose levels elicited strong humoral and T cell responses after the second dose. When administered with AS03, even the 3.75 μg CoVLP dose elicited neutralizing antibody titers that were ∼10-times higher than those observed in individuals recovering from Covid-19 as well as consistent and balanced IFN-γ and IL-4 responses. Although many CoVLP formulations were immunogenic, in the absence of established correlates of protection and given the advantages of dose-sparing in the context of the on-going pandemic, these findings suggest that CoVLP (3.75 μg)+AS03 has a good benefit/risk ratio and support the transition of this formulation to studies in expanded populations and to efficacy evaluationsShorter AbstractBackgroundVirus-like particles (VLP) displaying recombinant SARS-CoV-2 spike protein trimers were produced by transient expression in Nicotiana benthamiana. This candidate vaccine (CoVLP) was evaluated in healthy adults 18-55 years of age alone or with AS03 or CpG1018 (NCT04450004).MethodsThis randomized, partially-blinded, two-dose, dose-escalation study assessed CoVLP (3.75, 7.5 or 15 µg/dose) administered intramuscularly alone or with CpG1018 or AS03 in SARS-CoV-2 seronegative adults (18-55 years). Primary endpoints of safety and immunogenicity are reported to day 42. Neutralizing antibodies (NtAb) were assessed using a VSV pseudovirus assay and cellular responses by ELISpot (IFNγ, IL-4).Results180 subjects (avg.34.3yrs) were recruited. All formulations were well-tolerated but adjuvants increased reactogenicity. Adverse events were highest in the CoVLP+AS03 groups and increased in frequency/severity after dose two. CoVLP alone elicited weak humoral responses but modest cellular responses were detectable after dose two. Both adjuvants increased immunogenicity significantly, particularly after dose two. A significant NtAb response after dose one was only seen in CoVLP+AS03 groups. The vaccine dose had little impact on levels of NtAb responses achieved in the CoVLP+AS03 groups. Both adjuvants also increased IFNγ and IL-4 responses but these cellular responses also tended to be highest in the AS03-adjuvanted groups.ConclusionCoVLP ± adjuvants was well-tolerated. Several adjuvanted formulations elicited strong humoral and T cell responses after dose 2. Even at the lowest CoVLP+AS03 dose, NtAb titers were ∼10-times higher than in convalescent serum with a balanced IFNγ and IL-4 response. These findings support the transition of CoVLP (3.75μg+AS03) to further clinical evaluation.Research In ContextEvidence before this studyThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was recognized as the causative agent of COVID-19 in early 2020. Since that time, >150 candidate vaccines are reported to be under development of which 47 have entered clinical trials (https://www.who.int/publications/m/item/draft-landscape-of-covid-19-candidate-vaccines accessed Nov 4, 2020). No vaccine to prevent COVID-19 has been licensed yet for either emergency or general use in North America or Europe. We searched PubMed for research articles published between July 2019 and November 4, 2020, using the terms “SARS-CoV-2”, “vaccine”, “clinical trial” OR “human”, AND “phase”. The same terms were used to search ClinTrials.gov. No language restrictions were applied. We identified 10 peer-reviewed studies, describing phase 1 or 1/2 trials using a range of novel (eg: RNA, DNA, non-replicating virus vectored) and more traditional vaccine approaches (eg: inactivated virus or recombinant protein ± adjuvants). None of these candidate vaccines was produced in plants. These reports demonstrate that several different vaccination strategies (typically delivered in two doses 14-28 days apart) are capable of eliciting neutralizing antibody responses. In several cases, vaccine-induced cellular responses against SARS-COV-2 antigens - predominantly the spike (S) protein - can also be demonstrated. Although local and systemic adverse events following vaccination have varied between reports, the trials published to date suggest that each of these candidate vaccines is well-tolerated in the context of an evolving pandemic.Added value of this studyWe report the results of the first clinical study of CoVLP, a virus-like particle (VLP) vaccine that is produced by transient transfection of Nicotiana benthamiana plants. These VLPs spontaneously assemble at the plant cell membrane and display SARS-COV-2 trimers of stabilized pre-fusion S protein on their surface. The vaccine was administered as two intramuscular doses 21 days apart at three dose levels (S protein content 3.75, 7,5 or 15μg) alone or adjuvanted with either CpG1018 or AS03. All formulations were well-tolerated although both adjuvants increased reactogenicity. Humoral (anti-S IgG and neutralizing antibodies) as well as cellular responses (IFNg and IL4 ELISpots) were detectable in almost all subjects who received adjuvanted formulations 21 days after the second dose at all COVLP dose levels. Both antibody and cellular responses were highest in subjects who received AS03-adjuvanted formulations. Even at the lowest dose level (3.75μg), the neutralizing antibody titers 21 days after the second dose in subjects who received the AS03-afdjuvanted vaccine were 10-50-fold higher than those seen in subjects recovering from COVID-19 infection.Implications of all the available evidenceEffective vaccines against SARS-CoV-2 are urgently needed to reduce the burden of disease and contribute to ending the global pandemic. Although no immune correlates for SARS-CoV-2 have been defined, it is likely that both arms of the immune system contribute to protection. After two doses of CoVLP (3.75μg+AS03), strong humoral and cellular responses were induced supporting the further clinical development of this vaccine.

Partial neutralization of the Golgi lumen pH by the ectopic expression of influenza virus M2 proton channel is useful to stabilize acid-labile recombinant proteins in plant cells, but the impact of pH gradient mitigation on host cellular functions has not been investigated. Here, we assessed the unintended effects of M2 expression on the leaf proteome of

The ability to express heterologous proteins in plants has led to the concept of using plants as "bioreactors" or "biofactories" for the production of pharmaceutical proteins. Although initial studies were promising, the pathway to commercialization and deployment in a clinical setting has proven to be a somewhat rocky road. This Review examines the technical developments that have led to the current increase in interest in the use of plants for the production of pharmaceutical proteins, particularly in the context of clinical trials.

Partial neutralization of the Golgi lumen pH by ectopic expression of influenza virus M2 proton channel stabilizes acid-labile and protease-susceptible recombinant proteins in the plant cell secretory pathway. Here, we assessed the impact of M2 channel expression on the proteome ofNicotiana benthamianaleaf tissue infiltrated with the bacterial gene vectorAgrobacterium tumefaciens, keeping in mind the key role of pH homeostasis on secreted protein processing and the involvement of protein secretion processes in plant cells upon microbial challenge. The proteomes of leaves agroinfiltrated with an empty vector or with an M2 channel-encoding vector were compared with the proteome of non-infiltrated leaves using a iTRAQ quantitative proteomics procedure. Leaves infiltrated with the empty vector had a low soluble protein content compared to non-infiltrated leaves, associated with a strong decrease of photosynthesis-associated proteins (including Rubisco) and a parallel increase of stress-related secreted proteins (including pathogenesis-related proteins, protease inhibitors and molecular chaperones). M2 expression partly compromised these alterations of the proteome to restore original soluble protein and Rubisco contents, associated with higher levels of translation-associated (ribosomal) proteins and reduced levels of stress-related proteins in the apoplast. Proteome changes in M2-expressing leaves were determined both transcriptionally and post-transcriptionally, to alter the steady-state levels of proteins not only along the secretory pathway but also in other cellular compartments including the chloroplast, the cytoplasm, the nucleus and the mitochondrion. These data illustrate the cell-wide influence of Golgi lumen pH homeostasis on the leaf proteome ofN. benthamianaplants responding to microbial challenge. They underline in practice the relevance of carefully considering the eventual off-target effects of accessory proteins used to modulate specific cellular or metabolic functions in plant protein biofactories.

Summary Cellular engineering approaches have been proposed to mitigate unintended proteolysis in plant protein biofactories, involving the design of protease activity‐depleted environments by gene silencing or in situ inactivation with accessory protease inhibitors. Here, we assessed the impact of influenza virus M2 proton channel on host protease activities and recombinant protein processing in the cell secretory pathway of Nicotiana benthamiana leaves. Transient co‐expression assays with M2 and GFP variant pHluorin were first conducted to illustrate the potential of proton export from the Golgi lumen to promote recombinant protein yield. A fusion protein‐based system involving protease‐sensitive peptide linkers to attach inactive variants of tomato cystatin SlCYS8 was then designed to relate the effects of M2 on protein levels with altered protease activities in situ. Secreted versions of the cystatin fusions transiently expressed in leaf tissue showed variable ‘fusion to free cystatin’ cleavage ratios, in line with the occurrence of protease forms differentially active against the peptide linkers in the secretory pathway. Variable ratios were also observed for the fusions co‐expressed with M2, but the extent of fusion cleavage was changed for several fusions, positively or negatively, as a result of pH increase in the Golgi. These data indicating a remodelling of endogenous protease activities upon M2 expression confirm that the stability of recombinant proteins in the plant cell secretory pathway is pH‐dependent. They suggest, in practice, the potential of M2 proton channel to modulate the stability of protease‐susceptible secreted proteins in planta via a pH‐related, indirect effect on host resident proteases.

A key factor influencing the yield of biopharmaceuticals in plants is the ratio of recombinant to host proteins in crude extracts. Postextraction procedures have been devised to enrich recombinant proteins before purification. Here, we assessed the potential of methyl jasmonate (MeJA) as a generic trigger of recombinant protein enrichment in Nicotiana benthamiana leaves before harvesting. Previous studies have reported a significant rebalancing of the leaf proteome via the jasmonate signalling pathway, associated with ribulose 1,5-bisphosphate carboxylase oxygenase (RuBisCO) depletion and the up-regulation of stress-related proteins. As expected, leaf proteome alterations were observed 7 days post-MeJA treatment, associated with lowered RuBisCO pools and the induction of stress-inducible proteins such as protease inhibitors, thionins and chitinases. Leaf infiltration with the Agrobacterium tumefaciens bacterial vector 24 h post-MeJA treatment induced a strong accumulation of pathogenesis-related proteins after 6 days, along with a near-complete reversal of MeJA-mediated stress protein up-regulation. RuBisCO pools were partly restored upon infiltration, but most of the depletion effect observed in noninfiltrated plants was maintained over six more days, to give crude protein samples with 50% less RuBisCO than untreated tissue. These changes were associated with net levels reaching 425 μg/g leaf tissue for the blood-typing monoclonal antibody C5-1 expressed in MeJA-treated leaves, compared to less than 200 μg/g in untreated leaves. Our data confirm overall the ability of MeJA to trigger RuBisCO depletion and recombinant protein enrichment in N. benthamiana leaves, estimated here for C5-1 at more than 2-fold relative to host proteins.

Summary Biopharmaceutical glycoproteins produced in plants carry N-glycans with plant-specific residues core α(1,3)-fucose and β(1,2)-xylose, which can significantly impact the activity, stability and immunogenicity of biopharmaceuticals. In this study, we have employed sequence-specific transcription activator-like effector nucleases (TALENs) to knock out two α(1,3)-fucosyltransferase (FucT) and the two β(1,2)-xylosyltransferase (XylT) genes within Nicotiana benthamiana to generate plants with improved capacity to produce glycoproteins devoid of plant-specific residues. Among plants regenerated from N. benthamiana protoplasts transformed with TALENs targeting either the FucT or XylT genes, 50% (80 of 160) and 73% (94 of 129) had mutations in at least one FucT or XylT allele, respectively. Among plants regenerated from protoplasts transformed with both TALEN pairs, 17% (18 of 105) had mutations in all four gene targets, and 3% (3 of 105) plants had mutations in all eight alleles comprising both gene families; these mutations were transmitted to the next generation. Endogenous proteins expressed in the complete knockout line had N-glycans that lacked β(1,2)-xylose and had a significant reduction in core α(1,3)-fucose levels (40% of wild type). A similar phenotype was observed in the N-glycans of a recombinant rituximab antibody transiently expressed in the homozygous mutant plants. More importantly, the most desirable glycoform, one lacking both core α(1,3)-fucose and β(1,2)-xylose residues, increased in the antibody from 2% when produced in the wild-type line to 55% in the mutant line. These results demonstrate the power of TALENs for multiplexed gene editing. Furthermore, the mutant N. benthamiana lines provide a valuable platform for producing highly potent biopharmaceutical products.

International audience; Influenza virus‐like particles (VLPs) have been shown to induce a safe and potent immune response through both humoral and cellular responses. They represent promising novel influenza vaccines. Plant‐based biotechnology allows for the large‐scale production of VLPs of biopharmaceutical interest using different model organisms, including Nicotiana benthamiana plants. Through this platform, influenza VLPs bud from the plasma membrane and accumulate between the membrane and the plant cell wall. To design and optimize efficient production processes, a better understanding of the plant cell wall composition of infiltrated tobacco leaves is a major interest for the plant biotechnology industry. In this study, we have investigated the alteration of the biochemical composition of the cell walls of N. benthamiana leaves subjected to abiotic and biotic stresses induced by the Agrobacterium‐mediated transient transformation and the resulting high expression levels of influenza VLPs. Results show that abiotic stress due to vacuum infiltration without Agrobacterium did not induce any detectable modification of the leaf cell wall when compared to non infiltrated leaves. In contrast, various chemical changes of the leaf cell wall were observed post‐Agrobacterium infiltration. Indeed, Agrobacterium infection induced deposition of callose and lignin, modified the pectin methylesterification and increased both arabinosylation of RG‐I side chains and the expression of arabinogalactan proteins. Moreover, these modifications were slightly greater in plants expressing haemagglutinin‐based VLP than in plants infiltrated with the Agrobacterium strain containing only the p19 suppressor of silencing.

Background Plant-made biotherapeutics are gathering momentum and some plant glycoproteins are allergens. Glycans with core β1-2xylose and α1,3fucose motifs and antennae terminated by mannose residues ( e.g. : MMXF) are found on several plant allergens and can cross-react with glyco-epitopes from other sources. To date, reactivity to these cross-reactive determinants has not been associated with clinical symptoms. Objective We produced VLP vaccines bearing the hemagglutinin(HA) of H5(A/Indonesia/5/05) or H1(A/California/07/09) influenza viruses by transfection of Nicotiana benthamiana . Subjects enrolled in Phase I/II trials were followed for evidence of allergy/hypersensitivity and development of antibodies against plant glyco-epitopes. Methods A total of 280/349 subjects received either one (H1) or 2 doses (H5) of vaccine (5–45 μg of HA/dose) intramuscularly including 40 with pre-existing plant allergies. Subjects were monitored for 6 months. IgG and IgE to plant glyco-epitopes were measured by ELISA using corn-/egg-derived avidin and bromelain as target antigens. Results No subject developed allergic/hypersensitivity symptoms. Some (34%) developed transient IgG and, in some cases IgE, to plant glyco-epitopes but no subject mounted an IgE response to the MMXF motif. Antibodies returned to baseline by 6 months in most subjects. Conclusion VLP vaccines bearing influenza HA glycoproteins can elicit transient IgG and, in some cases, IgE responses that are not associated with either the development or worsening of allergic/hypersensitivity symptoms.

We previously described a procedure for the use of plant protease inhibitors as "companion" accessory proteins to prevent unwanted proteolysis of clinically useful recombinant proteins in leaf crude protein extracts (Benchabane et al. Methods Mol Biol 483:265-273, 2009). Here we describe the use of these inhibitors for the protection of recombinant proteins in planta, before their extraction from leaf tissues. A procedure is first described involving inhibitors co-expressed along-and co-migrating-with the protein of interest in host plant cells. An alternative, single transgene scheme is then described involving translational fusions of the recombinant protein and companion inhibitor. These approaches may allow for a significant improvement of protein steady-state levels in leaves, comparable to yield improvements observed with protease-deficient strains of less complex protein expression hosts such as E. coli or yeasts.

Recombinant proteins face major constraints along the plant cell secretory pathway, including proteolytic processing compromising their structural integrity. Here, we demonstrate the potential of protease inhibitors as in situ stabilizing agents for recombinant proteins migrating towards the leaf apoplast. Genomic data for Arabidopsis, rice and Nicotiana spp. were assessed to determine the relative incidence of protease families in the cell secretory pathway. Transient expression assays with the model platform Nicotiana benthamiana were then performed to test the efficiency of protease inhibitors in stabilizing proteins targeted to the apoplast. Current genomic data suggest the occurrence of proteases from several families along the secretory pathway, including A1 and A22 Asp proteases; C1A and C13 Cys proteases; and S1, S8 and S10 Ser proteases. In vitro protease assays confirmed the presence of various proteases in N. benthamiana leaves, notably pointing to the deposition of A1- and S1-type activities preferentially in the apoplast. Accordingly, transient expression and secretion of the A1/S1 protease inhibitor, tomato cathepsin D inhibitor (SlCDI), negatively altered A1 and S1 protease activities in this cell compartment, while increasing the leaf apoplast protein content by ~45% and improving the accumulation of a murine diagnostic antibody, C5-1, co-secreted in the apoplast. SlCYS9, an inhibitor of C1A and C13 Cys proteases, had no impact on the apoplast proteases and protein content, but stabilized C5-1 in planta, presumably upstream in the secretory pathway. These data confirm, overall, the potential of protease inhibitors for the in situ protection of recombinant proteins along the plant cell secretory pathway. © 2011 The Authors. Plant Biotechnology Journal

During the last decade, the spectre of an influenza pandemic of avian origin has led to a revision of national and global pandemic preparedness plans and has stressed the need for more efficient influenza vaccines and manufacturing practices. The 2009 A/H1N1 (swine flu) outbreak has further emphasized the necessity to develop new solutions for pandemic influenza vaccines. Influenza virus-like particles (VLPs)-non-infectious particles resembling the influenza virus-represent a promising alternative to inactivated and split-influenza virions as antigens, and they have shown uniqueness by inducing a potent immune response through both humoral and cellular components of the immune system. Our group has developed a plant-based transient influenza VLP manufacturing platform capable of producing influenza VLPs with unprecedented speed. Influenza VLP expression and purification technologies were brought to large-scale production of GMP-grade material, and pre-clinical studies have demonstrated that low doses of purified, plant-produced influenza VLPs induce a strong and broad immune response in mice and ferrets. This review positions the recent developments towards the successful production of influenza VLPs in plants, including the production of VLPs from other human viruses and other forms of influenza antigens. The platform developed for large-scale production of VLPs is also presented along with an assessment of the speed of the platform to produce the first experimental vaccine lots from the identification of a new influenza strain.

International audience; Plant-based transient expression is potentially the most rapid and cost-efficient system for the production of recombinant pharmaceutical proteins, but safety concerns associated with plant-specific N-glycosylation have hampered its adoption as a commercial production system. In this article, we describe an approach based on the simultaneous transient co-expression of an antibody, a suppressor of silencing and a chimaeric human β 1,4-galactosyltransferase targeted for optimal activity to the early secretory pathway in agroinfiltrated Nicotiana benthamiana leaves. This strategy allows fast and high-yield production of antibodies with human-like N-glycans and, more generally, provides solutions to many critical problems posed by the large-scale production of therapeutic and vaccinal proteins, specifically yield, volume and quality.

A strain-specific vaccine represents the best possible response to the threat of an influenza pandemic. Rapid delivery of such a vaccine to the world's population before the peak of the first infection wave seems to be an unattainable goal with the current influenza vaccine manufacturing capacity. Plant-based transient expression is one of the few production systems that can meet the anticipated surge requirement. To assess the capability of plant agroinfiltration to produce an influenza vaccine, we expressed haemagglutinin (HA) from strains A/Indonesia/5/05 (H5N1) and A/New Caledonia/20/99 (H1N1) by agroinfiltration of Nicotiana benthamiana plants. Size distribution analysis of protein content in infiltrated leaves revealed that HA was predominantly assembled into high-molecular-weight structures. H5-containing structures were purified and examination by transmission electron microscopy confirmed virus-like particle (VLP) assembly. High-performance thin layer chromatography analysis of VLP lipid composition highlighted polar and neutral lipid contents comparable with those of purified plasma membranes from tobacco plants. Electron microscopy of VLP-producing cells in N. benthamiana leaves confirmed that VLPs accumulated in apoplastic indentations of the plasma membrane. Finally, immunization of mice with two doses of as little as 0.1 microg of purified influenza H5-VLPs triggered a strong immune response against the homologous virus, whereas two doses of 0.5 microg of H5-VLPs conferred complete protection against a lethal challenge with the heterologous A/Vietnam/1194/04 (H5N1) strain. These results show, for the first time, that plants are capable of producing enveloped influenza VLPs budding from the plasma membrane; such VLPs represent very promising candidates for vaccination against influenza pandemic strains.

We previously described a procedure for the use of plant protease inhibitors as "companion" accessory proteins to prevent unwanted proteolysis of clinically useful recombinant proteins in leaf crude protein extracts (Benchabane et al. Methods Mol Biol 483:265-273, 2009). Here we describe the use of these inhibitors for the protection of recombinant proteins in planta, before their extraction from leaf tissues. A procedure is first described involving inhibitors co-expressed along-and co-migrating-with the protein of interest in host plant cells. An alternative, single transgene scheme is then described involving translational fusions of the recombinant protein and companion inhibitor. These approaches may allow for a significant improvement of protein steady-state levels in leaves, comparable to yield improvements observed with protease-deficient strains of less complex protein expression hosts such as E. coli or yeasts.

A promoter fusion (Sh35) combining upstream regulatory regions from the maize Sh1 promoter with a truncated 35S promoter, Δ9035 (–90 to +8) has been compared with the original Sh1 promoter for its capacity to promote expression of the β-glucuronidase (GUS) gene in stably transformed tomato plants. For both promoters, very faint GUS expression was detected in the vegetative tissues, and no expression was detected in the fruit pericarp tissues. However, in the seed, Sh1 promoted low GUS expression but Sh35 directed 25-fold higher GUS expression. For both constructs, the profile of GUS expression was similar to that of endogenous sucrose synthase activity, but maximal GUS activity was reached 15 days after the peak of sucrose synthase activity.

The expression of clinically useful proteins in plants has been bolstered by the development of high-yielding systems for transient protein expression using agroinfiltration. There is a need now to know more about how host plant development and metabolism influence the quantity and quality of recombinant proteins. Endogenous proteolysis is a key determinant of the stability and yield of recombinant proteins in plants. Here we characterised cysteine (C1A) and aspartate (A1) protease profiles in leaves of the widely used expression host Nicotiana benthamiana, in relation with the production of a murine IgG, C5-1, targeted to the cell secretory pathway. Agroinfiltration significantly altered the distribution of C1A and A1 proteases along the leaf age gradient, with a correlation between leaf age and the level of proteolysis in whole-cell and apoplast protein extracts. The co-expression of tomato cystatin SlCYS8, an inhibitor of C1A proteases, alongside C5-1 increased antibody yield by nearly 40% after the usual 6-days incubation period, up to ∼3 mg per plant. No positive effect of SlCYS8 was observed in oldest leaves, in line with an increased level of C1A protease activity and a very low expression rate of the inhibitor. By contrast, C5-1 yield was greater by an additional 40% following 8- to 10-days incubations in younger leaves, where high SlCYS8 expression was maintained. These findings confirm that the co-expression of recombinant protease inhibitors is a promising strategy for increasing recombinant protein yields in plants, but that further opportunity exists to improve this approach by addressing the influence of leaf age and proteases of other classes.

Recombinant proteins face major constraints along the plant cell secretory pathway, including proteolytic processing compromising their structural integrity. Here, we demonstrate the potential of protease inhibitors as in situ stabilizing agents for recombinant proteins migrating towards the leaf apoplast. Genomic data for Arabidopsis, rice and Nicotiana spp. were assessed to determine the relative incidence of protease families in the cell secretory pathway. Transient expression assays with the model platform Nicotiana benthamiana were then performed to test the efficiency of protease inhibitors in stabilizing proteins targeted to the apoplast. Current genomic data suggest the occurrence of proteases from several families along the secretory pathway, including A1 and A22 Asp proteases; C1A and C13 Cys proteases; and S1, S8 and S10 Ser proteases. In vitro protease assays confirmed the presence of various proteases in N. benthamiana leaves, notably pointing to the deposition of A1- and S1-type activities preferentially in the apoplast. Accordingly, transient expression and secretion of the A1/S1 protease inhibitor, tomato cathepsin D inhibitor (SlCDI), negatively altered A1 and S1 protease activities in this cell compartment, while increasing the leaf apoplast protein content by ∼45% and improving the accumulation of a murine diagnostic antibody, C5-1, co-secreted in the apoplast. SlCYS9, an inhibitor of C1A and C13 Cys proteases, had no impact on the apoplast proteases and protein content, but stabilized C5-1 in planta, presumably upstream in the secretory pathway. These data confirm, overall, the potential of protease inhibitors for the in situ protection of recombinant proteins along the plant cell secretory pathway.

During the last decade, the spectre of an influenza pandemic of avian origin has led to a revision of national and global pandemic preparedness plans and has stressed the need for more efficient influenza vaccines and manufacturing practices. The 2009 A/H1N1 (swine flu) outbreak has further emphasized the necessity to develop new solutions for pandemic influenza vaccines. Influenza virus-like particles (VLPs)-non-infectious particles resembling the influenza virus-represent a promising alternative to inactivated and split-influenza virions as antigens, and they have shown uniqueness by inducing a potent immune response through both humoral and cellular components of the immune system. Our group has developed a plant-based transient influenza VLP manufacturing platform capable of producing influenza VLPs with unprecedented speed. Influenza VLP expression and purification technologies were brought to large-scale production of GMP-grade material, and pre-clinical studies have demonstrated that low doses of purified, plant-produced influenza VLPs induce a strong and broad immune response in mice and ferrets. This review positions the recent developments towards the successful production of influenza VLPs in plants, including the production of VLPs from other human viruses and other forms of influenza antigens. The platform developed for large-scale production of VLPs is also presented along with an assessment of the speed of the platform to produce the first experimental vaccine lots from the identification of a new influenza strain.

The improvements in agroinfiltration methods for plant-based transient expression now allow the production of significant amounts of recombinant proteins in a matter of days. While vacuum-based agroinfiltration has been brought to large scale to meet the cost, speed and surge capacity requirements for vaccine and therapeutic production, the more accessible and affordable syringe agroinfiltration procedure still represents a fast and high-yielding approach to recombinant protein production at lab scale. The procedure exemplified here has proven its reproducibility and high-yield capacity for the production of proteins with varying levels of complexity, including monoclonal antibodies.

The improvements in agroinfiltration methods for plant-based transient expression now allow the production of significant amounts of recombinant proteins in a matter of days. While vacuum-based agroinfiltration has been brought to large scale to meet the cost, speed and surge capacity requirements for vaccine and therapeutic production, the more accessible and affordable syringe agroinfiltration procedure still represents a fast and high-yielding approach to recombinant protein production at lab scale. The procedure exemplified here has proven its reproducibility and high-yield capacity for the production of proteins with varying levels of complexity, including monoclonal antibodies.

Compared with other plant expression systems used for pharmaceutical protein production, alfalfa offers the advantage of very homogeneous N-glycosylation. Therefore, this plant was selected for further attempts at glycoengineering. Two main approaches were developed in order to humanize N-glycosylation in alfalfa. The first was a knock-down of two plant-specific N-glycan maturation enzymes, beta1,2-xylosyltransferase and alpha1,3-fucosyltransferases, using sense, antisense and RNA interference strategies. In a second approach, with the ultimate goal of rebuilding the whole human sialylation pathway, human beta1,4-galactosyltransferase was expressed in alfalfa in a native form or in fusion with a targeting domain from N-acetylglucosaminyltransferase I, a glycosyltransferase located in the early Golgi apparatus in Nicotiana tabacum. Both knock-down and knock-in strategies strongly, but not completely, inhibited the biosynthesis of alpha1,3-fucose- and beta1,2-xylose-containing glycoepitopes in transgenic alfalfa. However, recombinant human beta1,4-galactosyltransferase activity in transgenic alfalfa completely prevented the accumulation of the Lewis a glycoepitope on complex N-glycans.

International audience; Development of convenient strategies for identification of plant N-glycan profiles has been driven by the emergence of plants as an expression system for therapeutic proteins. In this article, we reinvestigated qualitative and quantitative aspects of plant N-glycan profiling. The extraction of plant proteins through a phenol/ammonium acetate procedure followed by deglycosylation with peptide N-glycosidase A (PNGase A) and coupling to 2-aminobenzamide provides an oligosaccharide preparation containing reduced amounts of contaminants from plant cell wall polysaccharides. Such a preparation was also suitable for accurate qualitative and quantitative evaluation of the N-glycan content by mass spectrometry. Combining these approaches allows the profiling to be carried out from as low as 500 mg of fresh leaf material. We also demonstrated that collision-induced dissociation (CID) mass spectrometry in negative mode of N-glycans harboring alpha(1,3)- or alpha(1,6)-fucose residue on the proximal GlcNAc leads to specific fragmentation patterns, thereby allowing the discrimination of plant N-glycans from those arising from mammalian contamination.

The use of multiple copies of vectors based on either full-length or deleted versions of cowpea mosaic virus RNA-2 for the production of heteromeric proteins in plants was investigated. Co-infiltration of two full-length RNA-2 constructs containing different marker genes into Nicotiana benthamiana in the presence of RNA-1 showed that the two foreign proteins were efficiently expressed within the same cell in inoculated tissue. Furthermore, the proteins were co-localized to the same subcellular compartments, an essential prerequisite for heteromer formation. However, segregation of two separate RNA-2 molecules, and therefore expression of the two proteins, was observed on systemic spread of the recombinant viruses. Thus, efficient assembly of heteromeric proteins is likely to occur only in inoculated tissue. To determine the optimum approach for expression in inoculated tissue, the heavy and light chains of the blood group-typing immunoglobulin G (IgG) C5-1 were inserted into full-length and deleted versions of RNA-2, and the constructs were agroinfiltrated in the presence of RNA-1. The results obtained showed that full-size IgG molecules accumulated using both approaches, but that the levels were significantly higher when deleted RNA-2 vectors were used. The levels were also greatly enhanced by the inclusion of an endoplasmic reticulum retention signal at the C-terminus of the heavy chain. As the potential benefit of using full-length RNA-2 constructs, the ability to spread systemically, appears to be irrelevant to the production of heteromeric proteins, the use of deleted versions of RNA-2 is clearly advantageous, particularly as they offer the benefit of biocontainment.

Plants are a low-cost and contamination-free factory for the production of recombinant pharmaceutical proteins. However, plant-made pharmaceuticals differ from their mammalian homologues by the structure of their N-linked glycans. For instance, most mammalian glycoproteins harbour terminal sialic acids that control their half-life in the bloodstream. The absence of the whole sialylation machinery in plants is of major concern as non-sialylated plant-made pharmaceuticals may not perform at their full potential in humans, because of their removal from the circulation through the involvement of hepatic cell receptors. In this context, we have investigated the synthesis of N-acetylneuraminic acid (Neu5Ac) in the cytosol of plants by either the re-routing of the endogenous 3-deoxy-d-manno-2-octulosonic acid (Kdo) biosynthetic pathway or the expression of microbial Neu5Ac-synthesizing enzymes. In this paper, we demonstrate that the plant Kdo-8P synthase is not able to use N-acetyl d-mannosamine as a substrate, and thus re-routing of the Kdo pathway for the synthesis of Neu5Ac is not possible. Consequently, we expressed genes encoding Neu5Ac lyase from Escherichia coli and Neu5Ac synthase (neuB2) from Campylobacter jejuni in plants. These resulted in the production of functional enzymes in the cytosol, which in turn can catalyse the synthesis of Neu5Ac in vitro. Experiments were carried out on two models, Bright Yellow 2 (BY2) tobacco cells and Medicago sativa (alfalfa), the perennial legume crop.

In 1986, it was shown that tobacco plants and sunflower calluses could express recombinant human growth hormone as a fusion protein [1]. Since then, a diverse range of plant systems has been used for the production of pharmaceuticals [2, 3]. We have developed a production system based on the leaves of alfalfa (Medicago sativa L.), a choice made originally because of the plant’s many favorable agronomic characteristics. Alfalfa is a perennial plant, so vegetative growth can be maintained for many years. For molecular farming, this characteristic, combined with the ease of clonal propagation through stem cutting, makes alfalfa a robust bioreactor with regard to batch-to-batch reproducibility. Among perennial plants, legume forage crops such as alfalfa have the advantage of fixing atmospheric nitrogen, thus reducing the need for fertilizers. Moreover, as a feed fodder crop, alfalfa has benefited from important research aiming to increase leaf protein content, so that today’s varieties produce as much as 30 mg total protein per gram fresh weight. In addition to these appealing agronomic characteristics, biotechnological research has revealed additional benefits for the production of pharmaceuticals in alfalfa. Expression cassettes have been optimized for protein expression in alfalfa leaves. Methods for transient protein expression have been developed so that it is now possible to use agroinfiltration or the transformation of protoplasts for earlystage demonstration and validation steps. In addition, glycosylation studies have shown that alfalfa is capable of producing recombinant glycoproteins with homogenous (uniform) glycosylation patterns. This chapter provides an overview of the tools that have been developed and optimized specifically for the production of pharmaceuticals in alfalfa, with the emphasis on recent technological breakthroughs. The ability of alfalfa leaves to produce complex recombinant proteins of pharmaceutical interest is discussed and illustrated with recent data obtained in our laboratories. Data are presented concerning the production and characterization of alfalfa-derived C5-1, a diagnostic anti-human 1

The role of sucrose synthase (SuSy) in tomato fruit was studied in transgenic tomato (Lycopersicon esculentum) plants expressing an antisense fragment of fruit-specific SuSy RNA (TOMSSF) under the control of the cauliflower mosaic virus 35S promoter. Constitutive expression of the antisense RNA markedly inhibited SuSy activity in flowers and fruit pericarp tissues. However, inhibition was only slight in the endosperm and was undetectable in the embryo, shoot, petiole, and leaf tissues. The activity of sucrose phosphate synthase decreased in parallel with that of SuSy, but acid invertase activity did not increase in response to the reduced SuSy activity. The only effect on the carbohydrate content of young fruit was a slight reduction in starch accumulation. The in vitro sucrose import capacity of fruits was not reduced by SuSy inhibition at 23 days after anthesis, and the rate of starch synthesized from the imported sucrose was not lessened even when SuSy activity was decreased by 98%. However, the sucrose unloading capacity of 7-day-old fruit was substantially decreased in lines with low SuSy activity. In addition, the SuSy antisense fruit from the first week of flowering had a slower growth rate. A reduced fruit set, leading to markedly less fruit per plant at maturity, was observed for the plants with the least SuSy activity. These results suggest that SuSy participates in the control of sucrose import capacity of young tomato fruit, which is a determinant for fruit set and development.